Fig. 3: The N-terminal IDR of SLBP engages the UPF1 helicase core via direct protein–protein interactions. | Nature Communications

Fig. 3: The N-terminal IDR of SLBP engages the UPF1 helicase core via direct protein–protein interactions.

From: Mechanistic insights into recruitment and regulation of the RNA helicase UPF1 in replication-dependent histone mRNA decay

Fig. 3: The N-terminal IDR of SLBP engages the UPF1 helicase core via direct protein–protein interactions.The alternative text for this image may have been generated using AI.

A Mapping the SLBP-binding site on UPF1. GST-pulldown assays were performed with GST-UPF1 proteins and SLBPfl. GST-GYF serves as a negative control. The top and bottom panels show the inputs and bound fractions (precipitate) in this and all subsequent GST-pulldown experiments. The helicase core of UPF1 is sufficient for its interaction with SLBP. All GST-pulldown assays in this study were performed independently three times with similar results. B Mapping the binding site for UPF1 on SLBP. Top: schematic representation of the domain arrangement of human SLBP and the SLBP variants tested. Bottom: GST-pulldown using GST-UPF1-Hel and SLBP proteins. The N-terminal IDR of SLBP is necessary for binding to UPF1. A complete gel including negative controls with GST-GYF is shown in Supplementary Fig. 3A. C Analytical size exclusion chromatography (SEC) of UPF1-Hel and SLBP-N in the absence (gray traces) and presence (black traces) of SL26 RNA. PAGE analyses corresponding to the SEC runs are shown on the right (top: with SL26, bottom: without SL26). The SL RNA was visualized by ethidium bromide (Et-Br) staining of the urea-PAGE gel. Binding of SL26 RNA to SLBP-N stabilizes the protein. The experiment was performed twice with similar results. D Linkage map showing the interlinks between UPF1 and SLBP, derived from crosslinking-mass spectrometry analysis of the UPF1-Hel:SLBP-N:U-SL complex (see Supplementary Fig. 3E) using BS3. The analysis was performed in triplicate on the same sample. Interlinks are depicted as black or gray lines, where linewidth corresponds to the count of peptides identified for each crosslink. Intralinks within each protein are shown as gray arcs, represented according to peptide count, as for the interlinks. The schematics represent the protein variants used for reconstitution of the complex and are colored according to Figs. 1A and 3B. Positions of all lysine residues in each protein are indicated by black vertical lines in the schematic. See also Supplementary Fig. 3F for mapping of UPF1-SLBP crosslinked residues onto the UPF1:U-SL RNA and SLBP:SL26 RNA structures. Mass spectrometry data are presented in Supplementary Table 2.

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